Activation of the Ghrelin Receptor is Described by a Privileged Collective Motion: A Model for Constitutive and Agonist-induced Activation of a Sub-class A G-Protein Coupled Receptor (GPCR) |
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Authors: | Nicolas Floquet Céline M'Kadmi Didier Gagne Jacky Marie Jean-Claude Galleyrand Jean Martinez |
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Institution: | 1 Institut des Biomolécules Max Mousseron (I.B.M.M.), CNRS UMR5247 - Université Montpellier 1 - Université Montpellier 2, Faculté de Pharmacie, 15 avenue Charles Flahault, B.P. 14 491, 34093 Montpellier Cedex 5, France 2 Institut de Biochimie et Biophysique Moléculaire et Cellulaire, UMR8619, Bât. 430, Université Paris-Sud, 91405 Orsay, France |
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Abstract: | Three homology models of the human ghrelin receptor (GHS-R1a) have been generated from the available X-ray structures of rhodopsin (RHO model), opsin (OPS model) and beta-2 adrenergic receptor (B2 model). The latter was used as a starting point for combined molecular dynamics simulation (MDS) and full atom normal modes analysis (NMA). A low-frequency normal mode (mode 16) perfectly reproduced the intracellular motions observed between B2 and RHO models; in the opposite direction along the same mode, the generated structures are closer to the OPS model, suggesting a direct link with GHS-R1a activation. This was in agreement with motions of the seven transmembranous segments, increase of the solvent accessibility of the 140-ERY-142 sequence, and flip of the Trp276 (C
WLP) residue, some features related to GPCRs activation. According to our model, His280 was proposed to stabilize Trp276 in the active state; this was verified by site-directed mutagenesis and biochemical characterization of the resulting H280A and H280S mutants, which were fully functional but sharing an important decrease of their basal activities. Docking performed with short ghrelin derivatives Gly-Ser-Ser
octa]-Phe-NH
2 and Gly-Ser-Ser
octa]-Phe-Leu-NH
2 allowed the identification of a robust position of these peptides in the active site of the receptor. This model was refined by MDS and validated by docking experiments performed on a set of 55 ghrelin receptor ligands based on the 1,2,4- triazole scaffold. Finally, NMA performed on the obtained peptide-receptor complex suggested stabilization of the Trp276 residue and of the whole receptor in the active state, preventing the motion observed along mode 16 computed for the unbound receptor. Our results show that NMA offers a powerful approach to study the conformational diversity and the activation mechanism of GPCRs. |
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Keywords: | GHS-R1a growth hormone secretagogue receptor 1a MDS molecular dynamics simulation NMA normal mode analysis GPCR G protein-coupled receptor 7TM seven transmembranous MRMS mass weighted root mean square RMSD root-mean-square deviation GHR4 peptide GSSF-NH2 GHR5 peptide GSSFL-NH2 Sub-P substance P analogue WT wild type |
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